The invention relates to peptides comprising an immunogenic site of poliovirus and DNA fragments containing nucleotide sequences coding for these peptides. The invention also relates to vaccinating principles bringing such peptides into play, these principles being adapted to induce in the host, man or animal, the production of antibodies active not only against themselves, but also against complete infectious polioviruses.
In French Patent Application No. 82 02013 filed 8 Feb. 1982 there have already been described DNA fragments coding for an immunogenic peptide capable of inducing in vivo the synthesis of antipoliovirus antibodies. These DNA fragments possess a length not exceeding that of a DNA fragment comprising of the order of 1.2 kb (kilo-pairs of bases). These fragments are more particularly characterized in that they contain an nucleotide sequence coding for the protein VP-1, which has been found to bear essential antigenic determinants brought into play at the level of the immunogenicity of the corresponding infectious poliovirus. In fact, this peptide is capable of forming antigen-antibody complexes with monoclonal or polyclonal neutralizing serums obtained from animals in which whole poliovirus had been injectd (serum of D-specificity).
DNA type sequences coding for immunogenic peptides of the above-indicated type are illustrated in the succession of the appended FIGS. 1 and 2, for one of them, and in the succession of FIGS. 3 and 4, also appended, for another DNA fragment containing the abovesaid sequence. The locations of certain restriction sites to which reference will be made below are also indicated in these drawings The numbering of the successive nucleotides taking part in the constitution of these DNAs is effected from the 5' end. With respect to the constitution of the clonable DNA of the poliovirus from which the abovesaid DNAs have been obtained, reference will be made to the article of Sylvie VAN DER WERF and other authors, entitled "Molecular Cloning of the Genome of Poliovirus" in Proc. Nat. Acad. Sci. USA, Vol. 78, No. 10, pp. 59-83, 59-87, October 1981.
The invention arises from the discovery that peptides corresponding to the DNA sequences contained in the preceding ones, but much smaller than the latter, carried nonetheless antigenic determinants enabling their use in the constitution of vaccinating principles effective against the corresponding polioviruses. From the peptides concerned, some can be isolated the size of which is sufficiently small for them to be directly accessible by chemical synthesis.
The invention provides in addition technique enabling the determination, within DNAs of relatively large size which form the subject of French Patent Application No. 82 02013, of those of the smaller DNA sequences to which correspond peptides having determinants or antigenic sites making them suitable for use in the production of vaccinating principles against corresponding whole and infectious polioviruses.
In this regard, the longest of the DNA sequences according to the invention is constituted by the fragment bounded at its opposite end by XbaI sites located in the regions defined by the positions 2546 and 2861 of FIG. 1.
The invention relates more particularly still to those of the DNA sequences contained within the preceeding one and which code a peptide capable of being recognized by monoclonal antibodies active both against "C" and "D" particles originating from a same poliovirus and against the structural polypeptide VP-1 of the capsid of the same poliovirus. It is this type of monoclonal antibody which is concerned in all circumstances in the description which follows, except when it is otherwise specified.
Such antibodies are obtained from hybridoma which have been obtained by the carrying out of the fusion of spleen cells of an animal previously immunized by a virus or virion having a "C" antigenicity (obtained by thermal treatment for 1 hour at 56.degree. C. of the corresponding infectious poliovirus having "D" antigenicity) and suitable myelomatous cells using a method known per se, by the cultivation of the clones or hybrid cells obtained and by the selection of the clones which are found to produce monoclonal antibodies active both against the virus with "C" antigenicity, the homologous infection viruses (virions) with "D" antigenicity and against the corresponding protein VP-1. The homologous virions contemplated herein are advantageously of the 1-type (Mahoney). Such monoclonal antibodies (denoted hereafter under the expression "CD-VP-1 antibodies (or "C3")), the hybrid cells capable of producing them and a process for their production were described in French Patent Application No. 82 19338 filed on 18 Nov. 1982. Two of the cell hybrids formed have been deposited at the National Culture Collection of Micro-Organisms of the Pasteur Institute of Paris (C.N.C.M.), respectively under No. I-208 and No. I-209.
This sequence according to the invention has the following structure:
TCT AGA GAC GCT CTC CCA AAC ACT GAA GCC AGT GGA CCA ACA CAC TCC AAG GAA ATT CCG GCA CTC ACC GCA GTG GAA ACT GGG GCC ACA AAT CCA CTA GTC CCT TCT GAT ACA GTG CAA ACC AGA CAT GTT GTA CAA CAT AGG TCA AGG TCA GAG TCT AGC ATA GAG TCT TTC TTC GCG CGG GGT GCA TGC GTG ACC ATT ATG ACC GTG GAT AAC CCA GCT TCC ACC ACG AAT AAG CAT AAG CTA TTT GCA GTG TGG AAG ATC ACT TAT AAA GAT ACT GTC CAG TTA CGG AGG AAA TTG GAG TTC TTC ACC TAT TCT. PA0 Ser Arg Asp Ala Leu Pro Asn Thr Glu Ala Ser Gly Pro Thr His Ser Lys Glu Ile Pro Ala Leu Thr Ala Val Glu Thr Gly Ala Thr Asn Pro Leu Val Pro Ser Asp Thr Val Gln Thr Arg His Val Val Gln His Arg Ser Arg Ser Glu Ser Ser Ile Glu Ser Phe Phe Ala Arg Gly Ala Cys Val Thr Ile Met Thr Val Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp Lys Leu Phe Ala Val Trp Lys Ile Thr Tyr Lys Asp Thr Val Gln Leu Arg Arg Lys Leu Glu Phe Phe Thr Tyr Ser
The invention also relates to any DNA sequence coding for a peptide having immunogenic properties similar to those of the peptide coded by the abovesaid nucleotide sequence. In particular any triplet of the sequence can be replaced, either by a distinct triplet coding for the same amino acid or for a distinct amino acid, to the extent that the substitution of the second for the first in the peptide coded by the DNA sequence concerned, will not fundamentally alter the immunogenic properties of the peptide coded by the so modified DNA sequence. In particular, the invention relates to any DNA sequence of this type coding for a peptide which can be recognized by the above C3 antibody.
The invention also relates to any nucleotide sequence of smaller length contained in the preceding one, as soon as it codes for a peptide still also capable of being recognized by the C3 antibody.
Among the DNA sequences comprised within the scope of the invention, are included those containing nucleotide sequences coding for the peptide sequence His 65-Phe 105 defined below, and more particularly for the nucleotide sequence 2671-2792 of the gene coding for the polypeptide of VP-1 structure of the poliovirus of FIG. 1.
Other preferred DNA sequences within the field of the invenjion are those which code for the peptide sequences His 65-Ile110 defined below, and more particularly again the nucleotide sequence Pro 95-Ile110 from the same gene.
The invention relates naturally to the polypeptides containing the peptide sequences coded by the above-said DNA sequences. It relates in particular to the sequence of formula:
The invention also relates to any peptide having equivalent immunogenic properties under the conditions which have already been indicated with respect to the peptides coded by the DNA sequences defined above. In this respect the invention relates more particularly to the following sequence, called below "His 65-Phe 105 sequence".
______________________________________ His Val Val Gln His Arg Ser Arg Ser Glu Ser Ser Ile Glu Ser 70 Phe Phe Ala Arg Gly Ala Cys Val Thr Ile 80 Met Thr Val Asp Asn Pro Ala Ser Thr Thr 90 Asn Lys Asp Lys Leu Pne 100 ______________________________________
or called below "sequence H is 65-Ile 110".
______________________________________ His Val Val Gln His Arg Ser Arg Ser Glu Ser Ser Ile Glu Ser 70 Phe Phe Ala Arg Gly Ala Cys Val Thr Ile 80 Met Thr Val Asp Asn Pro Ala Ser Thr Thr 90 Asn Lys Asp Lys Leu Phe Ala Val Trp Lys 100 Ile 110 ______________________________________
The invention relates more particularly also to those of the peptides which contain the following peptide sequence, called below ASP 93-Leu 104: Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp Lys Leu.
The invention relates naturally also to the vectors, particularly of the plasmid or phage type, containing an insert formed by anyone of the DNA sequences such as have been defined above. These modified vectors may be employed in the transformation of cellular organisms or of suitable microorganisms, in order to induce the production by the latter of polypeptides, possibly hybrid ones, containing a peptide sequence recognizable by the CD-PV1 or C3 monoclonal antibodies or other antibodies recognizing the infectious virus. These polypeptides, possibly hybrid ones, also form part of the invention.
The invention provides a process enabling the identification, within a DNA sequence normally contained within the DNA of a determined poliovirus, of those of the smaller sequences which are capable of coding for an immunogenic peptide or capable of being utilized in the manufacture of an immunogen principle enabling the production of antibodies active against the corresponding whole poliovirus.
This process is essentially charaterized in that, starting from a plasmid containing an insert formed of an initial sequence recognized as presumably containing a smaller sequence capable of coding for an immunogenic peptide or a peptide likely of being part of an immunogenic principle, one linearizes said plasmid at the level of a restriction site external to said smaller sequence, one trims the linearized plasmid in controlled manner with an exonucleolytic enzyme, such as enzyme Bal 31, one recircularizes the trimmed plasmid with a DNA ligase, one transforms a suitable microorganism transformable by the corresponding plasmid and capable of expresing the insert contained in the latter, and one detects the possible presence of a peptide liable of bearing the immunogenic site of the type concerned among the expression products of said microorganisms, by contacting said expression products with a monoclonal CD-PV1 antibody, said cycle of operations which has been defined being repeated until the disappearance of the detection of said immunogenic peptide among the expression products of the micro-organism as transformed by the last recircularized plasmid.
It is possible, at the end of each of the cycles of the above-defined process, for example, by comparison of the restriction maps of the plasmid before and after the abovesaid trimming operation, to determine those of the DNA sequences which have been removed between two successive trims and, consequently, when the possibility of detection of an immunogenic peptide under the aboveindicated conditions ceases, to correlate this result with one of the sequences eliminated in the course of the preceding trimming operation, this eliminated DNA sequence participating in the coding for said immunogenic peptide. The structure of the eliminated sequence (or of the eliminated sequences), may of course result of determinations of terminal nucleotide sequences, before and after the trimming concerned respectively.